This application claims benefit of PCT/DE98/00879, filed Mar. 26, 1998, now abandoned, which claims benefit of DE 197 12 899.8, filed Mar. 27, 1997, which issued as German Patent No. 197 12 899 on Dec. 10, 1998.
The invention relates to a process for recombinantly preparing picornavirus particles, in particular hepatitis A virus particles, their precursors and particles which are derived therefrom.
It is known from the state of the art to prepare picornavirus strains by culturing in eukaryotic cells. Such culturing is particularly inefficient in the case of particular members of the Picornaviridae family, for example the hepatitis A virus. A further disadvantage is that the picornaviruses which are cultured in the eukaryotic cells are infectious. They have to be chemically inactivated before being used, for example as a dead vaccine. Residues of the substance used for the inactivation, for example formaldehyde, may elicit allergic reactions and alter the antigenicity of the virus particle.
Stapelton, J. T. et al. 1990 have described (in Hollinger, F. B. et al.: Viral hepatitis and liver disease, 50-54) a more efficient method which can be used, in particular, for preparing empty, recombinant hepatitis A virus particles. The particle yields which are achieved by this method are likewise not adequate for commercial applications.
It is furthermore known from the state of the art that polyprotein processing is mediated by 3CD, 3C and 2A in the case of the enterovirus and rhinovirus genera, by L-proteinase and 3C in the case of the cardiovirus and apthovirus genera, and by 3C in the case of the hepatovirus genus. It is not known from the state of the art whether other nonstructural proteins are involved in the formation of empty virus particles.
The object of the present invention is to specify a process which eliminates the disadvantages of the state of the art. The aim is, in particular, to make available a more efficient process.
According to the invention, a structural protein precursor molecule and the corresponding P3 region (3ABCD) are coexpressed, in cis or in trans, for the purpose of recombinantly preparing picornavirus particles, in particular hepatitis A virus particles, their precursors and particles which are derived therefrom. The part proteins 2B and 2C are advantageously not expressed in this connection. One of the advantages of the novel process is that the inhibitory effects associated with expressing the part proteins 2B and 2C are eliminated and use is only made of the minimum sequences which are required for efficiently synthesizing empty picornavirus particles. This improves the efficiency of the process, on the one hand, and excludes the possibility of picornaviral replication, on the other hand. Using the complete P3 region markedly increases the efficiency of the synthesis of empty picornavirus particles, or their precursor molecules, as compared with only using the 3C and/or 3CD proteinases.
Advantageously, the structural protein precursor molecule possesses sequences encoding P1 or P1-2A. The P1 structural protein precursor molecule is in particular used in the case of the enterovirus and rhinovirus genera. In the case of the cardiovirus, aphthovirus and hepatovirus genera, use is preferably made of the structural protein precursor molecule which possesses the sequence encoding P1-2A.
A particularly efficient preparation of empty hepatitis A virus particles can be achieved when the VP1-2A cleavage site at amino acid position 273/274 is occupied by a glutamate/serine pair or a glutamine/serine pair, with the indication of the amino acid position relating to the sequence of the attenuated strain HM175 (in accordance with Cohen, J. I. et al. 1987, Journal of Virology 61:3035-3039).
When the structural protein precursor molecule and the P3 proteins are expressed in cis, there is equimolar expression of the two genome regions. This enables empty hepatitis A virus particles to be prepared efficiently. An even more efficient preparation of hepatitis A virus particles can be achieved by optimizing the molar ratio of the structural protein precursor molecule and the P3 proteins by means of expressing in trans. The coexpression in trans can be carried out by coexpressing the structural protein precursor molecule and the P3 region when they are encoded on separate vectors. This coexpression can also be carried out using a bicistronic expression vector or a bicistronic mRNA. In this context, at least two promoter sequences or translation initiation sequences which are of differing efficiencies are advantageously located in front of the P3 cistron and the structural protein cistron. This makes it possible to adjust the molar ratio of the structural protein precursor molecule and the corresponding P3 region optimally for achieving efficient synthesis of empty picornavirus particles.
It has proved to be advantageous for the expression of the proteins of the P3 region to be controlled negatively in cis by means of cloning sequence elements of the 5xe2x80x2 non-translated region (=5xe2x80x2 NTR) in front of the sequences encoding the P3 region. The negative control of the expression of the proteins of the P3 region contributes, when this region is being coexpressed together with the structural protein precursor molecule, to the two components being expressed in an optimal ratio.
The negatively controlling sequence elements employed can expediently be the sequences which are located in front of the internal ribosome binding site of the 5xe2x80x2 NTR. Particularly in the case of the hepatitis A virus, it has proved to be advantageous for the sequence elements of the 5xe2x80x2 NTR to be the 24 or 45 5xe2x80x2 terminal nucleotides of the 5xe2x80x2 NTR of the hepatitis A virus genome, or to be derived therefrom.
According to another organizational feature of the process, the coexpression is carried out in eukaryotic or prokaryotic cells or in the corresponding cell extracts, with the sequences which are to be coexpressed being cloned 3xe2x80x2 of a eukaryotic or of a prokaryotic promoter. The Semliki forest virus expression system (Liljestrxc3x6m P., Garoff H., Bio/Technology 9, 1356-1361) or the baculovirus expression system (Bishop D H L, 1990, 62-67, Current Opinion in Biotechnology) can, for example, be used in this context.
The invention furthermore encompasses a kit or a combination of means for carrying out the above-described process. Finally, the picornavirus particles according to the invention can be used for preparing a medicament or vaccine or for preparing galenics or diagnostic agents.